Fibroblast growth factor 14

ABSTRACT

Disclosed is a human Fibroblast growth factor-14 polypeptide and DNA(RNA) encoding such polypeptide. Also provided is a procedure for producing such polypeptide by recombinant techniques. Also disclosed are methods for utilizing such polypeptide for promoting wound healing for example as a result of burns and ulcers, to prevent neuronal damage due to associated with stroke and promote neuronal growth, and to prevent skin aging and hair loss, to stimulate angiogenesis, mesodermal induction in early embryos and limb regeneration. Antagonists against such polypeptides and their use as a therapeutic to prevent abnormal cellular proliferation, hyper-vascular diseases and epithelial lens cell proliferation are also disclosed. Diagnostic methods for detecting mutations in the coding sequence and alterations in the concentration of the polypeptides in a sample derived from a host are also disclosed.

[0001] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptide ofthe present invention have been putatively identified as fibroblastgrowth factor/heparin binding growth factor, hereinafter referred to as“FGF-14”. The invention also relates to inhibiting the action of suchpolypeptides.

[0002] Fibroblast growth factors are a family of proteins characteristicof binding to heparin and are, therefore, also called heparin bindinggrowth factors (HBGF). Expression of different members of these proteinsare found in various tissues and are under particular temporal andspatial control. These proteins are potent mitogens for a variety ofcells of mesodermal, ectodermal, and endodemal origin, includingfibroblasts, corneal and vascular endothelial cells, granulocytes,adrenal cortical cells, chondrocytes, myoblasts, vascular smooth musclecells, lens epithelial cells, melanocytes, keratinocytes,oligodendrocytes, astrocytes, osteoblasts, and hematopoietic cells.

[0003] Each member has functions overlapping with others and also hasits unique spectrum of functions. In addition to the ability tostimulate proliferation of vascular endothelial cells, both FGF-1 and 2are chemotactic for endothelial cells and FGF-2 has been shown to enableendothelial cells to penetrate the basement membrane. Consistent withthese properties, both FGF-1 and 2 have the capacity to stimulateangiogenesis. Another important feature of these growth factors is theirability to promote wound healing. Many other members of the FGF familyshare similar activities with FGF-1 and 2 such as promoting angiogenesisand wound healing. Several members of the FGF family have been shown toinduce mesoderm formation and to modulate differentiation of neuronalcells, adipocytes and skeletal muscle cells.

[0004] Other than these biological activities in normal tissues, FGFproteins have been implicated in promoting tumorigenesis in carcinomasand sarcomas by promoting tumor vascularization and as transformingproteins when their expression is deregulated.

[0005] The FGF family presently consists of eight structurally-relatedpolypeptides: basic FGF, acidic FGF, int 2, hst 1/k-FGF, FGF-5, FGF-6,keratinocyte growth factor, AIGF (FGF-8) and recently a glia-activatingfactor has been shown to be a novel heparin-binding growth factor whichwas purified from the culture supernatant of a human glioma cell line(Miyamoto, M. et al., Mol. and Cell. Biol., 13(7):4251-4259 (1993). Thegenes for each have been cloned and sequenced. Two of the members, FGF-1and FGF-2, have been characterized under many names, but most often asacidic and basic fibroblast growth factor, respectively. The normal geneproducts influence the general proliferation capacity of the majority ofmesoderm and neuroectoderm-derived cells. They are capable of inducingangiogenesis in vivo and may play important roles in early development(Burgess, W. H. and Maciag, T., Annu. Rev. Biochem., 58:575-606,(1989)).

[0006] Many of the above-identified members of the FGF family also bindto the same receptors and elicit a second message through binding tothese receptors.

[0007] A eukaryotic expression vector encoding a secreted form of FGF-1has been introduced by gene transfer into porcine arteries. This modeldefines gene function in the arterial wall in vivo. FGF-1 expressioninduced intimal thickening in porcine arteries 21 days after genetransfer (Nabel, B. G., et al., Nature, 362:844-6 (1993)). It hasfurther been demonstrated that basic fibroblast growth factor mayregulate glioma growth and progression independent of its role in tumorangiogenesis and that basic fibroblast growth factor release orsecretion may be required for these actions (Morrison, R. S., et al., J.Neurosci. Res., 34:502-9 (1993)).

[0008] Fibroblast growth factors, such as basic FGF, have further beenimplicated in the growth of Kaposi's sarcoma cells in vitro (Huang, Y.Q., et al., J. Clin. Invest., 91:1191-7 (1993)). Also, the cDNA sequenceencoding human basic fibroblast growth factor has been cloned downstreamof a transcription promoter recognized by the bacteriophage T7 RNApolymerase. Basic fibroblast growth factors so obtained have been shownto have biological activity indistinguishable from human placentalfibroblast growth factor in mitogenicity, synthesis of plasminogenactivator and angiogenesis assays (Squires, C. H., et al., J. Biol.Chem., 263:16297-302 (1988)).

[0009] U.S. Pat. No. 5,155,214 discloses substantially pure mammalianbasic fibroblast growth factors and their production. The amino acidsequences of bovine and human basic fibroblast growth factor aredisclosed, as well as the DNA sequence encoding the polypeptide of thebovine species.

[0010] Newly discovered FGF-9 has around 30% sequence similarity toother members of the FGF family. Two cysteine residues and otherconsensus sequences in family members were also well conserved in theFGF-9 sequence. FGF-9 was found to have no typical signal sequence inits N terminus like those in acidic and basic FGF. However, FGF-9 wasfound to be secreted from cells after synthesis despite its lack of atypical signal sequence FGF (Miyamoto, M. et al., Mol. and Cell. Biol.,13(7):4251-4259 (1993). Further, FGF-9 was found to stimulate the cellgrowth of oligodendrocyte type 2 astrocyte progenitor cells, BALB/c3T3,and PC-12 cells but not that of human umbilical vein endothelial cells(Naruo, K., et al., J. Biol. Chem., 268:2857-2864 (1993).

[0011] Basic FGF and acidic FGF are potent modulators of cellproliferation, cell motility, differentiation, and survival and act oncell types from ectoderm, mesoderm and endoderm. These two FGFs, alongwith KGF and AIGF, were identified by protein purification. However, theother four members were isolated as oncogenes., expression of which wasrestricted to embryogenesis and certian types of cancers. FGF-9 wasdemonstrated to be a mitogen against glial cells. Members of the FGFfamily are reported to have oncogenic potency. FGF-9 has showntransforming potency when transformed into BALB/c3T3 cells (Miyamoto,M., et al., Mol. Cell. Biol., 13(7):4251-4259 (1993).

[0012] Androgen induced growth factor (AIGF), also known as FGF-8, waspurified from a conditioned medium of mouse mammary carcinoma cells(SC-3) simulated with testosterone. AIGF is a distinctive FGF-likegrowth factor, having a putative signal peptide and sharing 30-40%homology with known members of the FGF family. Mammalian cellstransformed with AIGF shows a remarkable stimulatory effect on thegrowth of SC-3 cells in the absence of androgen. Therefore, AIGFmediates androgen-induced growth of SC-3 cells, and perhaps other cells,since it is secreted by the tumor cells themselves.

[0013] The polypeptide of the present invention has been putativelyidentified as a member of the FGF family as a result of amino acidsequence homology with other members of the FGF family.

[0014] In accordance with one aspect of the present invention, there areprovided novel mature polypeptides as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof. The polypeptides of the present invention are ofhuman origin.

[0015] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding the polypeptidesof the present invention, including mRNAs, DNAs, cDNAs, genomic DNA, aswell as antisense analogs thereof and biologically active anddiagnostically or therapeutically useful fragments thereof.

[0016] In accordance with still another aspect of the present invention,there are provided processes for producing such polypeptides byrecombinant techniques through the use of recombinant vectors, such ascloning and expression plasmids useful as reagents in the recombinantproduction of the polypeptides of the present invention, as well asrecombinant prokaryotic and/or eukaryotic host cells comprising anucleic acid sequence encoding a polypeptide of the present invention.

[0017] In accordance with a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for screening for agonistsand antagonists thereto and for therapeutic purposes, for example,promoting wound healing for example as a result of burns and ulcers, toprevent neuronal damage due to associated with stroke and promoteneuronal growth, and to prevent skin aging and hair loss, to stimulateangiogenesis, mesodermal induction in early embryos and limbregeneration.

[0018] In accordance with yet a further aspect of the present invention,there are provided antibodies against such polypeptides.

[0019] In accordance with yet another aspect of the present invention,there are provided antagonists against such polypeptides and processesfor their use to inhibit the action of such polypeptides, for example,in the treatment of cellular transformation, for example, tumors, toreduce scarring and treat hyper-vascular diseases.

[0020] In accordance with another aspect of the present invention, thereare provided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to a polynucleotide encodinga polypeptide of the present invention

[0021] In accordance with yet another aspect of the present invention,there are provided diagnostic assays for detecting diseases orsusceptibility to diseases related to mutations in a nucleic acidsequence of the present invention and for detecting over-expression ofthe polypeptides encoded by such sequences.

[0022] In accordance with another aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

[0023] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0024] The following drawings are meant only as illustrations ofspecific embodiments of the present invention and are not meant aslimitations in any manner.

[0025]FIG. 1 depicts the cDNA sequence and corresponding deduced aminoacid sequence of FGF-14. The initial 26 amino acid residues represent aputative leader sequence.

[0026] In accordance with one aspect of the present invention, there areprovided isolated nucleic acids molecules (polynucleotides) which encodefor the mature polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID NOS:2) or for the mature polypeptide encoded by the cDNAof the clone deposited as ATCC Deposit No. 97148 on May 12, 1995.

[0027] The polynucleotide encoding FGF-14 of this invention wasdiscovered initially in a cDNA library derived from human cerebellumtissue. It is structurally related to all members of the fibroblastgrowth factor family and contains an open reading frame encoding apolypeptide of 225 amino acids of which the first 26 amino acidsrepresent a putative signal sequence such that the mature polypeptidecomprises 199 amino acids. Among the top matches are: 1) 40 % identityand 61 % sequence similarity to human FGF-9 over a stretch of 126 aminoacids; 2) 40% identity and 61% similarity to rat FGF-9 over a region of126 amino acids; 3) 36% identity and 57% similarity with human KGF overa stretch of 148 amino acids.

[0028] The FGF/HBGF family signature, GXCLX (S, T, A, G) X6(D, E) CXFXEis conserved in the polypeptide of the present invention, (X means anyamino acid residue; (D,E) means either D or E residue; X6 means any 6amino acid residues).

[0029] The polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 (SEQ ID NOS: 1) or that of thedeposited clone or may be a different coding sequence, as a result ofthe redundancy or degeneracy of the genetic code, encodes the same,mature polypeptide as the DNA of FIG. 1, (SEQ ID NOS:1) or the depositedcDNA.

[0030] The polynucleotides which encodes for the mature polypeptide ofFIG. 1 (SEQ ID NOS:2) or for the mature polypeptides encoded by thedeposited cDNA(s) may include: only the coding sequence for the maturepolypeptide; the coding sequence for the mature polypeptide andadditional coding sequence such as a leader or secretory sequence or aproprotein sequence; the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature polypeptide.

[0031] Thus, the term “polynucleotide encoding a polypeptiden”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0032] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptides having the deduced aminoacid sequence of FIG. 1 (SEQ ID NOS:2) or the polypeptides encoded bythe cDNA(s) of the deposited clone(s). The variants of thepolynucleotide may be a naturally occurring allelic variant of thepolynucleotide or a non-naturally occurring variant of thepolynucleotide.

[0033] Thus, the present invention includes polynucleotides encoding thesame mature polypeptide as shown in FIG. 1 (SEQ ID NOS:2) or the samemature polypeptides encoded by the cDNA(s) of the deposited clone(s) aswell as variants of such polynucleotides which variants encode for afragment, derivative or analog of the polypeptide of FIG. 1 (SEQ IDNOS:2) or the polypeptides encoded by the cDNA(s) of the depositedclone(s). Such nucleotide variants include deletion variants,substitution variants and addition or insertion variants.

[0034] As hereinabove indicated, the polynucleotide may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIG. 1 (SEQ ID NOS:1) or of the coding sequence of thedeposited clone(s). As known in the art, an allelic variant is analternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptides.

[0035] The present invention also includes polynucleotides, wherein thecoding sequence for the mature polypeptides may be fused in the samereading frame to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

[0036] Thus, for example, the polynucleotides of the present inventionmay encode for a mature protein, or for a protein having a prosequenceor for a protein having both a prosequence and a presequence (leadersequence).

[0037] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0038] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0039] Fragments of the full length FGF-14 gene may be used as ahybridization probe for a cDNA library to isolate the full length geneand to isolate other genes which have a high sequence similarity to thegene or similar biological activity. Probes of this type preferably haveat least 30 bases and may contain, for example, 50 or more bases. Theprobe may also be used to identify a cDNA clone corresponding to a fulllength transcript and a genomic clone or clones that contain thecomplete FGF-14 gene including regulatory and promotor regions, exons,and introns. An example of a screen comprises isolating the codingregion of the FGF-14 gene by using the known DNA sequence to synthesizean oligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

[0040] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO:1) orthe deposited cDNA(s).

[0041] Alternatively, the polynucleotide may have at least 20 bases,preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which has anidentity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO:l, for example, for recoveryof the polynucleotide or as a diagnostic probe or as a PCR primer.

[0042] Thus, the present invention is directed to polynucleotides havingat least a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

[0043] The deposit(s) referred to herein will be maintained under theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the purposes of Patent Procedure. These deposits areprovided merely as a convenience and are not an admission that a depositis required under 35 U.S.C. §112. The sequence of the polynucleotidescontained in the deposited materials, as well as the amino acid sequenceof the polypeptides encoded thereby, are incorporated herein byreference and are controlling in the event of any conflict with thedescription of sequences herein. A license may be required to make, useor sell the deposited materials, and no such license is hereby granted.

[0044] The present invention further relates to an FGF polypeptide whichhas the deduced amino acid sequence of FIG. 1 (SEQ ID NOS:2) or whichhas the amino acid sequence encoded by the deposited cDNA(s), as well asfragments, analogs and derivatives of such polypeptides.

[0045] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide of FIG. 1 (SEQ ID NOS:2) or those encoded by thedeposited cDNA(s), means polypeptides which retains essentially the samebiological function or activity as such polypeptides. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

[0046] The polypeptides of the present invention may be recombinantpolypeptides, natural polypeptides or synthetic polypeptides, preferablyrecombinant polypeptides.

[0047] The fragment, derivative or analog of the polypeptide of FIG. 1(SEQ ID NOS:2) or that encoded by the deposited cDNA (s) may be (i) onein which one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

[0048] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0049] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0050] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least a70% identity) to the polypeptide of SEQ ID NO:2 and more preferably atleast a 90% similarity (more preferably at least a 90% identity) to thepolypeptide of SEQ ID NO:2 and still more preferably at least a 95%similarity (still more preferably at least a 95% identity) to thepolypeptide of SEQ ID NO:2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0051] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0052] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0053] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0054] Host cells may be genetically engineered (transduced ortransformed or transfected) with the vectors of this invention which maybe, for example, a cloning vector or an expression vector. The vectormay be, for example, in the form of a plasmid, a viral particle, apliage, etc. The engineered host cells can be cultured in conventionalnutrient media modified as appropriate for activating promoters,selecting transformants or amplifying the FGF genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

[0055] The polynucleotide of the present invention may be employed forproducing a polypeptide by recombinant techniques. Thus, for example,the polynucleotide sequence may be included in any one of a variety ofexpression vehicles, in particular vectors or plasmids for expressing apolypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus,and pseudorabies. However, any other vector or plasmid may be used aslong as they are replicable and viable in the host.

[0056] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease sites by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0057] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0058] In addition, the expression vectors preferably contain a gene toprovide a phenotypic trait for selection of transformed host cells suchas dihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

[0059] The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence,may be employed to transform an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Salmonellatyphimurium, Streptomyces; fungal cells, such as yeast; insect cells,such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COSor Bowes melanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0060] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a,pNH46a (Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, pRITS(Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, PSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0061] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0062] In a further embodiment, the present invention relates to hostcells containing the above-described construct. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, 1986)).

[0063] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0064] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (Cold SpringHarbor, N.Y., 1989), the disclosure of which is hereby incorporated byreference.

[0065] Transcription of a DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication origin,and adenovirus enhancers.

[0066] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation, initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0067] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation, initiation and termination signals inoperable reading phase with a functional promoter. The vector willcomprise one or more phenotypic selectable markers and an origin ofreplication to ensure maintenance of the vector and to, if desirable,provide amplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0068] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0069] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0070] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification. Microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

[0071] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 viralgenome, for example, SV40 origin, early promoter, .enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

[0072] The polypeptide of the present invention may be recovered andpurified from recombinant cell cultures by methods used heretofore,including ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxyapatite chromatography and lectin chromatography.Protein refolding steps can be used, as necessary, in completingconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed for final purification steps.

[0073] The polypeptide of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated with mammalian or other eukaryotic carbohydrates or may benon-glycosylated. Polypeptides of the invention may also include aninitial methionine amino acid residue.

[0074] The polypeptide of the present invention, as a result of theability to stimulate vascular endothelial cell growth, may be employedin treatment for stimulating re-vascularization of ischemic tissues dueto various disease conditions such as thrombosis, arteriosclerosis, andother cardiovascular conditions. These polypeptide may also be employedto stimulate angiogenesis and limb regeneration.

[0075] The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

[0076] The polypeptide of the present invention may also be employedstimulate neuronal growth and to treat and prevent neuronal damageassociated with stroke and which occurs in certain neuronal disorders orneuro-degenerative conditions such as Alzheimer's disease, Parkinson'sdisease, and AIDS-related complex. FGF-14 has the ability to stimulatechondrocyte growth, therefore, they may be employed to enhance bone andperiodontal regeneration and aid in tissue transplants or bone grafts.

[0077] The polypeptide of the present invention may be also be employedto prevent skin aging due to sunburn by stimulating keratinocyte growth.

[0078] The FGF-14 polypeptide may also be employed for preventing hairloss, since FGF family members activate hair-forming cells and promotesmelanocyte growth. Along the same lines, the polypeptides of the presentinvention may be employed to stimulate growth and differentiation ofhematopoietic cells and bone marrow cells when used in combination withother cytokines.

[0079] The FGF-14 polypeptide may also be employed to maintain organsbefore transplantation or for supporting cell culture of primarytissues.

[0080] The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

[0081] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA, manufacture of DNAvectors and for the purpose of providing diagnostics and therapeuticsfor the treatment of human disease.

[0082] This invention provides a method for identification of thereceptors for the polypeptides of the present invention. The genesencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting(Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the polypeptides, for example, NIH3T3cells which are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the the polypeptide of the presentinvention, after they have been labelled. The polypeptides can belabeled by a variety of means including iodination or inclusion of arecognition site for a site-specific protein kinase.

[0083] Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

[0084] As an alternative approach for receptor identification, thelabeled polypeptides can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE analysis and exposed to X-ray film. Thelabeled complex containing the receptors of the polypeptides can beexcised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

[0085] This invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and ³[H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of ³[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of ³[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

[0086] In another method, a mammalian cell or membrane preparationexpressing a receptor for a polypeptide of the present invention isincubated with a labeled polypeptide of the present invention in thepresence of the compound. The ability of the compound to enhance orblock this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction of acompound to be screened and the FGF-14 receptor is measured and theability of the compound to bind to the receptor and elicit a secondmessenger response is measured to determine if the compound is apotential agonist or antagonist. Such second messenger systems includebut are not limited to, cAMP guanylate cyclase, ion channels, tyrosinephosphorylation or phosphoinositide hydrolysis.

[0087] Examples of antagonist compounds include antibodies, or in somecases, oligonucleotides, which bind to the receptor for the polypeptideof the present invention but elicit no second messenger response or bindto the FGF-14 polypeptide itself. Alternatively, a potential antagonistmay be a mutant form of the polypeptide which binds to the receptors,however, no second messenger response is elicited and, therefore, theaction of the polypeptide is effectively blocked.

[0088] Another antagonist compound to the FGF-14 gene and gene productis an antisense construct prepared using antisense technology. Antisensetechnology can be used to control gene expression through triple-helixformation or antisense DNA or RNA, both of which methods are based onbinding of a polynucleotide to DNA or RNA. For example, the 5′ codingportion of the polynucleotide sequence, which encodes for the maturepolypeptides of the present invention, is used to design an antisenseRNA oligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix -see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervanet al., Science, 251: 1360 (1991)), thereby preventing transcription andthe production of the polypeptides of the present invention. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the polypeptide (Antisense—Okano,J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of the polypeptide.

[0089] Potential antagonist compounds also include small molecules whichbind to and occupy the binding site of the receptors thereby making thereceptor inaccessible to its polypeptide such that normal biologicalactivity is prevented. Examples of small molecules include, but are notlimited to, small peptides or peptide-like molecules.

[0090] Antagonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

[0091] The antagonists may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

[0092] The antagonists may also be employed to prevent the growth ofscar tissue during wound healing.

[0093] The antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

[0094] The polypeptides, agonists and antagonists of the presentinvention may be employed in combination with a suitable pharmaceuticalcarrier to comprise a pharmaceutical composition for parenteraladministration. Such compositions comprise a therapeutically effectiveamount of the polypeptide, agonist or antagonist and a pharmaceuticallyacceptable carrier or excipient. Such a carrier includes but is notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The formulation should suit the mode ofadministration.

[0095] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides, agonists and antagonists of the presentinvention may be employed in conjunction with other therapeuticcompounds.

[0096] The pharmaceutical compositions may be administered in aconvenient manner such as by the oral, topical, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 μg/kg body weight and in most cases they will be administeredin an amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.In the specific case of topical administration, dosages are preferablyadministered from about 0.1 μg to 9 mg per cm².

[0097] The polypeptide of the invention and agonist and antagonistcompounds which are polypeptides, may also be employed in accordancewith the present invention by expression of such polypeptide in vivo,which is often referred to as “gene therapy.”

[0098] Thus, for example, cells may be engineered with a polynucleotide(DNA or RNA) encoding for the polypeptide ex vivo, the engineered cellsare then provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, cells may be engineeredby procedures known in the art by use of a retroviral particlecontaining RNA encoding for the polypeptide of the present invention.

[0099] Similarly, cells may be engineered in vivo for expression of thepolypeptide in vivo, for example, by procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such methods should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retroviral particle, for example, an adenovirus, which may beused to engineer cells in vivo after combination with a suitabledelivery vehicle.

[0100] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0101] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol III, and β-actin promoters). Other viral promoters whichmay be employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0102] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0103] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein byreference in its entirety. The vector may transduce the packaging cellsthrough any means known in the art. Such means include, but are notlimited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0104] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0105] This invention is also related to the use of the genes of thepresent invention as part of a diagnostic assay for detecting diseasesor susceptibility to diseases related to the presence of mutations inthe nucleic acid sequences encoding the polypeptide of the presentinvention.

[0106] Individuals carrying mutations in a gene of the present inventionmay be detected at the DNA level by a variety of techniques. Nucleicacids for diagnosis may be obtained from a patient's cells, such as fromblood, urine, saliva, tissue biopsy and autopsy material. The genomicDNA may be used directly for detection or may be amplified enzymaticallyby using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior toanalysis. RNA or cDNA may also be used for the same purpose. As anexample, PCR primers complementary to the nucleic acid encoding apolypeptide of the present invention can be used to identify and analyzemutations. For example, deletions and insertions can be detected by achange in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled RNA or alternatively, radiolabeled antisense DNAsequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

[0107] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0108] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and SI protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0109] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0110] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0111] The present invention also relates to a diagnostic assay fordetecting altered levels of FGF-14 proteins in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of abnormal cellular proliferation, forexample, a tumor. Assays used to detect levels of protein in a samplederived from a host are well-known to those of skill in the art andinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis, ELISA assays and “sandwich” assay. An ELISA assay (Coligan, etal., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initiallycomprises preparing an antibody specific to an antigen to thepolypeptides of the present invention, preferably a monoclonal antibody.In addition a reporter antibody is prepared against the monoclonalantibody. To the reporter antibody is attached a detectable reagent suchas radioactivity, fluorescence or, in this example, a horseradishperoxidase enzyme. A sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein like bovine serum albumen. Next,the monoclonal antibody is incubated in the dish during which time themonoclonal antibodies attach to any polypeptides of the presentinvention attached to the polystyrene dish. All unbound monoclonalantibody is washed out with buffer. The reporter antibody linked tohorseradish peroxidase is now placed in the dish resulting in binding ofthe reporter antibody to any monoclonal antibody bound to the protein ofinterest.

[0112] Unattached reporter antibody is then washed out. Peroxidasesubstrates are then added to the dish and the amount of color developedin a given time period is a measurement of the amount of a polypeptideof the present invention present in a given volume of patient samplewhen compared against a standard curve.

[0113] A competition assay may be employed wherein antibodies specificto a polypeptide of the present invention are attached to a solidsupport and labeled FGF-13 and a sample derived from the host are passedover the solid support and the amount of label detected, for example byliquid scintillation chromatography, can be correlated to a quantity ofa polypeptide of the present invention in the sample.

[0114] A “sandwich” assay is similar to an ELISA assay. In a “sandwich”assay a polypeptide of the present invention is passed over a solidsupport and binds to antibody attached to a solid support. A secondantibody is then bound to the polypeptide of interest. A third antibodywhich is labeled and specific to the second antibody is then passed overthe solid support and binds to the second antibody and an amount canthen be quantified.

[0115] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphism's) are presently available formarking chromosomal location. The mapping of DNAs to chromosomesaccording to the present invention is an important first step incorrelating those sequences with genes associated with disease.

[0116] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0117] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0118] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

[0119] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. (Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0120] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0121] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0122] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0123] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0124] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0125] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0126] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0127] In order to facilitate understanding of the following examples,certain frequently occurring methods and/or terms will be described.

[0128] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0129] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0130] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980). “Oligonucleotides” refers to either a singlestranded polydeoxynucleotide or two complementary polydeoxynucleotidestrands which may be chemically synthesized. Such syntheticoligonucleotides have no 5′ phosphate and thus will not ligate toanother oligonucleotide without adding a phosphate with an ATP in thepresence of a kinase. A synthetic oligonucleotide will ligate to afragment that has not been dephosphorylated.

[0131] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0132] Unless otherwise stated, transformation was performed asdescribed by the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLE 1 Bacterial Expression and Purification of FGF-14 Protein

[0133] The DNA sequence encoding FGF-14 ATCC #97148, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′sequences of the processed protein (minus the signal peptide sequence)and the vector sequences 3′ to the gene. Additional nucleotidescorresponding to the gene are added to the 5′ and 3′ sequences. The 5′oligonucleotide primer has the sequence 5′GCCAGAGCATGCAGCGGCGCGTGTGTCCCCGC 3′ (SEQ ID NO:3) and contains an SphIrestriction enzyme site. The 3′ sequence 5′GCCAGAAGATCTGGGGGCAGGGGGACTGGAAGG 3′ (SEQ ID NO:4) containscomplementary sequences to a BglII site and is followed by 21nucleotides of FGF-14 coding sequence.

[0134] The restriction enzyme sites correspond to the restriction enzymesites on the bacterial expression vector pQE70 (Qiagen, Inc. Chatsworth,Calif. 91311). pQE-70 encodes antibiotic resistance (Amp^(f)), abacterial origin of replication (ori), an IPTG-regulatable promoteroperator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-70 was then digested with NcoI and BglII.The amplified sequences are ligated into pQE-70 and are inserted inframe with the sequence encoding for the histidine tag and the ribosomebinding site (RBS). The ligation mixture is then used to transform E.coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described inSambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold SpringLaboratory Press, (1989). M15/rep4 contains multiple copies of theplasmid pREP4, which expresses the lacI repressor and also conferskanamycin resistance (Kan^(f)). Transformants are identified by theirability to grow on LB plates and ampicillin/kanamycin resistant colonieswere selected. Plasmid DNA is isolated and confirmed by restrictionanalysis. Clones containing the desired constructs are grown overnight(O/N) in liquid culturein LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells are grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression. Cells are grownan extra 3 to 4 hours. Cells are then harvested by centrifugation. Thecell pellet is solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized FGF-14 is purified from thissolution by chromatography on a Nickel-Chelate column under conditionsthat allow for tight binding by proteins containing the 6-His tag(Hochuli, E. et al., J. Chromatography 411:177-184 (1984)). The proteinsare eluted from the column in 6 molar quanidine HCl pH 5.0 and for thepurpose of renaturation adjusted to 3 molar guanidine HCl, 100 mM sodiumphosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione(oxidized). After incubation in this solution for 12 hours the proteinsare dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 2 Cloning and Expression of FGF-14 Using the BaculovirusExpression System

[0135] The DNA sequence encoding the full length FGF-14 protein, ATCC #97148, is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene:

[0136] The FGF-14 5′ primer has the sequence 5′ CTAGTGGATCCCATCATGGCGGCGCTGGCCAGT 3′ (SEQ ID NO:5) for the pA2 vector and containsa BamHI restriction enzyme site (in bold) followed by 4 nucleotidesresembling an efficient signal for the initiation of translation ineukaryotic cells (Kozak, M., J. Mol. Biol., 196:947-950 (1987) which isjust behind the first 18 nucleotides of the gene (the initiation codonfor translation “ATG” is underlined). For the pA2gp vector the 5′ primerhas the sequence 5° CGACTGGATCCCCAGCCGCGCGTGTGTCCC 3′ (SEQ ID NO:6).

[0137] The 3′ primer has the sequence 5′ CGACTTCTAGAATCAGGGGGCAGGGGGACTGGA 3′ (SEQ ID NO:7) and contains the cleavage site for therestriction endonuclease XbaI (in bold) and 22 nucleotides complementaryto the 3′ non-translated sequence of the gene.

[0138] The amplified sequences are isolated from a 1% agarose gel usinga commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment is then digested with the respective endonucleasesand purified again on a 1t agarose gel. This fragment is designated F2.

[0139] The vector pA2gp (and pA2) (modifications of pVL941 vector,discussed below) is used for the expression of the proteins using thebaculovirus expression system (for review see: Summers, M. D. and Smith,G. E. 1987, A manual of methods for baculovirus vectors and insect cellculture procedures, Texas Agricultural Experimental Station Bulletin No.1555). This expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby the recognition sites for the restriction endonucleases BamHI andXbaI. The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusthe beta-galactosidase gene from E.coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pA2 such as pRG1, pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

[0140] The plasmid is digested with the restriction enzymes anddephosphorylated using calf intestinal phosphatase by procedures knownin the art. The DNA is then isolated from a 1% agarose gel using thecommercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).This vector DNA is designated V2.

[0141] Fragment F2 and the dephosphorylated plasmid V2 are ligated withT4 DNA ligase. E.coli DH5α cells are then transformed and bacteriaidentified that contained the plasmid (pBacFGF-14) using the respectiverestriction enzymes. The sequence of the cloned fragment are confirmedby DNA sequencing.

[0142] 5 μg of the plasmid pBacFGF-14 is co-transfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

[0143] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid is mixed ina sterile well of microtiter plates containing 50 μl of serum freeGrace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards10 μl Lipofectin plus 90 μl Grace's medium are added, mixed andincubated for 15 minutes at room temperature. Then the transfectionmixture is added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in 35 mm tissue culture plates with 1 ml Grace's medium withoutserum. The plates are rocked back and forth to mix the newly addedsolution. The plates are then incubated for 5 hours at 27° C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.The plates are put back into an incubator and cultivation continued at27° C. for four days.

[0144] After four days the supernatant is collected and plaque assaysperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0145] Four days after the serial dilution the virus is added to thecells and blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

[0146] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-FGF-14 at a multiplicity of infection (MOI) of 2. Sixhours later the medium is removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵S-methionine and 5 μCi³⁵S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 3 Expression of Recombinant FGF-14 in COS Cells

[0147] The expression of plasmids, FGF-14-HA derived from a vectorpcDNA3/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E.coli replication origin, 4) CMVpromoter followed by a polylinker region, an SV40 intron andpolyadenylation site. DNA fragments encoding the entire FGF-14 precursorand an HA tag fused in frame to the 3′ end is cloned into the polylinkerregion of the vector, therefore, the recombinant protein expression isdirected under the CMV promoter. The HA tag corresponds to an epitopederived from the influenza hemagglutinin protein as previously described(I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R.Lerner, 1984, Cell 37:767, (1984)). The infusion of HA tag to the targetprotein allows easy detection of the recombinant protein with anantibody that recognizes the HA epitope.

[0148] The plasmid construction strategy is described as follows:

[0149] The DNA sequence encoding FGF-14, ATCC # 97148, is constructed byPCR using two primers: the 5′ primer 5′ CTAGTGGATCCCATCATGGCGGCGCTGGCCAGT 3′ (SEQ ID NO:8) contains a BamHI sitefollowed by 18 nucleotides of coding sequence starting from theinitiation codon; the 3′ sequence 5′ GATTTACTCGAGGGGGGCAGGGGGACTGGA 3′(SEQ ID NO:9) contains complementary sequences to an XhoI site,translation stop codon, HA tag and the last 18 nucleotides of the FGF-14coding sequence (not including the stop codon). Therefore, the PCRproduct contains a BamHI site, coding sequence followed by HA tag fusedin frame, a translation termination stop codon next to the HA tag, andan XhoI site.

[0150] The PCR amplified DNA fragments and the vector, pcDNA3/Amp, aredigested with the respective restriction enzymes and ligated. Theligation mixture is transformed into E. coli strain SURE (available fromStratagene Cloning Systems, La Jolla, Calig. 92037) the transformedculture is plated on ampicillin media plates and resistant colonies areselected. Plasmid DNA is isolated from transformants and examined byrestriction analysis for the presence of the correct fragment. Forexpression of the recombinant FGF-14 COS cells are transfected with theexpression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T.Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, (1989)). The expression of the FGF-14-HA protein is detected byradiolabelling and immunoprecipitation method (E. Harlow, D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells are labelled for 8 hours with ³⁵S-cysteine two days posttransfection. Culture media is then collected and cells are lysed withdetergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1k SDS, 1% NP-40, 0.5%DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Bothcell lysate and culture media are precipitated with an HA specificmonoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGEgels.

EXAMPLE 5 Expression via Gene Therapy

[0151] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0152] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked bythe long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0153] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer containing an EcoRI site and the3′ primer having contains a HindIII site. Equal quantities of theMoloney murine sarcoma virus linear backbone and the EcoRI and HimdIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

[0154] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0155] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0156] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

[0157] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 17 679 base pairs nucleic acid single linear cDNA 1 ATG GCG GCG CTGGCC AGT AGC CTG ATC CGG CAG AAG CGG GAG GTC CGC 48 Met Ala Ala Leu AlaSer Ser Leu Ile Arg Gln Lys Arg Glu Val Arg -25 -20 -15 GAG CCC GGG GGCAGC CGG CCG GTG TCG GCG CAG CGG CGC GTG TGT CCC 96 Glu Pro Gly Gly SerArg Pro Val Ser Ala Gln Arg Arg Val Cys Pro -10 -5 1 5 CGC GGC ACC AAGTCC CTT TGC CAG AAG CAG CTC CTC ATC CTG CTG TCC 144 Arg Gly Thr Lys SerLeu Cys Gln Lys Gln Leu Leu Ile Leu Leu Ser 10 15 20 AAG GTG CGA CTG TGCGGG GGG CGG CCC GCG CGG CCG GAC CGC GGC CCG 192 Lys Val Arg Leu Cys GlyGly Arg Pro Ala Arg Pro Asp Arg Gly Pro 25 30 35 GAG CCT CAG CTC AAA GGCATC GTC ACC AAA CTG TTC TGC CGC CAG GGT 240 Glu Pro Gln Leu Lys Gly IleVal Thr Lys Leu Phe Cys Arg Gln Gly 40 45 50 TTC TAC CTC CAG GCG AAT CCCGAC GGA AGC ATC CAG GGC ACC CCA GAG 288 Phe Tyr Leu Gln Ala Asn Pro AspGly Ser Ile Gln Gly Thr Pro Glu 55 60 65 70 GAT ACC AGC TCC TTC ACC CACTTC AAC CTG ATC CCT GTG GGC CTC CGT 336 Asp Thr Ser Ser Phe Thr His PheAsn Leu Ile Pro Val Gly Leu Arg 75 80 85 GTG GTC ACC ATC CAG AGC GCC AAGCTG GGT CAC TAC ATG GCC ATG AAT 384 Val Val Thr Ile Gln Ser Ala Lys LeuGly His Tyr Met Ala Met Asn 90 95 100 GCT GAG GGA CTG CTC TAC AGT TCGCCG CAT TTC ACA GCT GAG TGT CGC 432 Ala Glu Gly Leu Leu Tyr Ser Ser ProHis Phe Thr Ala Glu Cys Arg 105 110 115 TTT AAG GAG TGT GTC TTT GAG AATTAC TAC GTC CTG TAC GCC TCT GCT 480 Phe Lys Glu Cys Val Phe Glu Asn TyrTyr Val Leu Tyr Ala Ser Ala 120 125 130 CTC TAC CGC CAG CGT CGT TCT GGCCGG GCC TGG TAC CTC GGC CTG GAC 528 Leu Tyr Arg Gln Arg Arg Ser Gly ArgAla Trp Tyr Leu Gly Leu Asp 135 140 145 150 AAG GAG GGC CAG GTC ATG AAGGGA AAC CGA GTT AAG AAG ACC AAG GCA 576 Lys Glu Gly Gln Val Met Lys GlyAsn Arg Val Lys Lys Thr Lys Ala 155 160 165 GCT GCC CAC TTT CTG CCC AAGCTC CTG GAG GTG GCC ATG TAC CAG GAG 624 Ala Ala His Phe Leu Pro Lys LeuLeu Glu Val Ala Met Tyr Gln Glu 170 175 180 CCT TCT CTC CAC AGT GTC CCCGAG GCC TCC CCT TCC AGT CCC CCT GCC 672 Pro Ser Leu His Ser Val Pro GluAla Ser Pro Ser Ser Pro Pro Ala 185 190 195 CCC TGAA 679 Pro 225 aminoacids amino acid linear protein 2 Met Ala Ala Leu Ala Ser Ser Leu IleArg Gln Lys Arg Glu Val Arg -26 -25 -20 -15 Glu Pro Gly Gly Ser Arg ProVal Ser Ala Gln Arg Arg Val Cys Pro -10 -5 1 5 Arg Gly Thr Lys Ser LeuCys Gln Lys Gln Leu Leu Ile Leu Leu Ser 10 15 20 Lys Val Arg Leu Cys GlyGly Arg Pro Ala Arg Pro Asp Arg Gly Pro 25 30 35 Glu Pro Gln Leu Lys GlyIle Val Thr Lys Leu Phe Cys Arg Gln Gly 40 45 50 Phe Tyr Leu Gln Ala AsnPro Asp Gly Ser Ile Gln Gly Thr Pro Glu 55 60 65 70 Asp Thr Ser Ser PheThr His Phe Asn Leu Ile Pro Val Gly Leu Arg 75 80 85 Val Val Thr Ile GlnSer Ala Lys Leu Gly His Tyr Met Ala Met Asn 90 95 100 Ala Glu Gly LeuLeu Tyr Ser Ser Pro His Phe Thr Ala Glu Cys Arg 105 110 115 Phe Lys GluCys Val Phe Glu Asn Tyr Tyr Val Leu Tyr Ala Ser Ala 120 125 130 Leu TyrArg Gln Arg Arg Ser Gly Arg Ala Trp Tyr Leu Gly Leu Asp 135 140 145 150Lys Glu Gly Gln Val Met Lys Gly Asn Arg Val Lys Lys Thr Lys Ala 155 160165 Ala Ala His Phe Leu Pro Lys Leu Leu Glu Val Ala Met Tyr Gln Glu 170175 180 Pro Ser Leu His Ser Val Pro Glu Ala Ser Pro Ser Ser Pro Pro Ala185 190 195 Pro 32 base pairs nucleic acid single linear DNA (genomic) 3GCCAGAGCAT GCAGCGGCGC GTGTGTCCCC GC 32 33 base pairs nucleic acid singlelinear DNA (genomic) 4 GCCAGAAGAT CTGGGGGCAG GGGGACTGGA AGG 33 33 basepairs nucleic acid single linear DNA (genomic) 5 CTAGTGGATC CCATCATGGCGGCGCTGGCC AGT 33 30 base pairs nucleic acid single linear DNA (genomic)6 CGACTGGATC CCCAGCGGCG CGTGTGTCCC 30 33 base pairs nucleic acid singlelinear DNA (genomic) 7 CGACTTCTAG AATCAGGGGG CAGGGGGACT GGA 33 33 basepairs nucleic acid single linear DNA (genomic) 8 CTAGTGGATC CCATCATGGCGGCGCTGGCC AGT 33 30 base pairs nucleic acid single linear DNA (genomic)9 GATTTACTCG AGGGGGGCAG GGGGACTGGA 30 17 Amino acids amino acid linearprotein 10 Gly Xaa Leu Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Asp Cys Xaa PheXaa 5 10 15 Glu 17 Amino acids amino acid linear protein 11 Gly Xaa LeuXaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Asp Cys Xaa Phe Xaa 5 10 15 Glu 17 Aminoacids amino acid linear protein 12 Gly Xaa Leu Xaa Ala Xaa Xaa Xaa XaaXaa Xaa Asp Cys Xaa Phe Xaa 5 10 15 Glu 17 Amino acids amino acid linearprotein 13 Gly Xaa Leu Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Asp Cys Xaa PheXaa 5 10 15 Glu 17 Amino acids amino acid linear protein 14 Gly Xaa LeuXaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Glu Cys Xaa Phe Xaa 5 10 15 Glu 17 Aminoacids amino acid linear protein 15 Gly Xaa Leu Xaa Thr Xaa Xaa Xaa XaaXaa Xaa Glu Cys Xaa Phe Xaa 5 10 15 Glu 17 Amino acids amino acid linearprotein 16 Gly Xaa Leu Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Glu Cys Xaa PheXaa 5 10 15 Glu 17 Amino acids amino acid linear protein 17 Gly Xaa LeuXaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Glu Cys Xaa Phe Xaa 5 10 15 Glu

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a polynucleotide encoding thepolypeptide comprising amino acid −26 to amino acid 199 as set forth inSEQ ID NO:2; (b) a polynucleotide encoding the polypeptide comprisingamino acid 1 to amino acid 199 as set forth in SEQ ID no:2; (c) apolynucleotide capable of hybridizing to and which is at least 70%identical to the polynucleotide of (a) or (b); and (d) a polynucleotidefragment of the polynucleotide of (a), (b) or (c).
 2. The polynucleotideof claim 1 wherein the polynucleotide is DNA.
 3. The polynucleotide ofclaim 1 comprising from nucleotide 1 to nucleotide 680 as set forth inSEQ ID NO:
 1. 4. The polynucleotide of claim 1 comprising fromnucleotide 79 to nucleotide 680 as set forth in SEQ ID NO:1.
 5. Anisolated polynucleotide comprising a member selected from the groupconsisting of: (a) a polynucleotide encoding a mature polypeptideencoded by the DNA contained in ATCC Deposit No. 97148; (b) apolynucleotide encoding the polypeptide expressed by the DNA containedin ATCC Deposit No. 97148; (c) a polynucleotide capable of hybridizingto and which is at least 70% identical to the polynucleotide of (a) or(b); and (d) a polynucleotide fragment of the polynucleotide of (a), (b)or (c).
 6. A vector containing the DNA of claim
 2. 7. A host cellgenetically engineered with the vector of claim
 6. 8. A process forproducing a polypeptide comprising: expressing from the host cell ofclaim 7 the polypeptide encoded by said DNA.
 9. A process for producingcells capable of expressing a polypeptide comprising geneticallyengineering cells with the vector of claim
 6. 10. A polypeptidecomprising a member selected from the group consisting of (i) apolypeptide having the deduced amino acid sequence of SEQ ID NO:2 andfragments, analogs and derivatives thereof; and (ii) a polypeptideencoded by the cDNA of ATCC Deposit No. 97148 and fragments, analogs andderivatives of said polypeptide.
 11. An antibody against the polypeptideof claim
 10. 12. A compound which inhibits the biological actions of thepolypeptide of claim
 10. 13. A compound which activates a receptor tothe polypeptide of claim
 10. 14. A method for the treatment of a patienthaving need of an FGF-14 polypeptide comprising: administering to thepatient a therapeutically effective amount of the polypeptide of claim10.
 15. A method for,the treatment of a patient having need to inhibitan FGF-14 polypeptide comprising: administering to the patient atherapeutically effective amount of the compound of claim
 12. 16. Themethod of claim 14 wherein said therapeutically effective amount of saidpolypeptide is administered by providing to the patient DNA encodingsaid polypeptide and expressing said polypeptide in vivo.
 17. The methodof claim 15 wherein said compound is a polypeptide and a therapeuticallyeffective amount of the compound is administered by providing to thepatient DNA encoding said antagonist and expressing said antagonist invivo.
 18. A process for identifying compounds active as agonists to thepolypeptide of claim 10 comprising: (a) combining a compound to bescreened and a reaction mixture containing cells under conditions wherethe cells are normally stimulated by said polypeptide, said reactionmixture containing a label incorporated into the cells as theyproliferate; and (b) determining the extent of proliferation of thecells to identify if the compound is an effective agonist.
 19. A processfor identifying compounds active as antagonists to the polypeptide ofclaim 10 comprising: (a) combining a compound to be screened, thepolypeptide and a reaction mixture containing cells under conditionswhere the cells are normally stimulated by said polypeptide, saidreaction mixture containing a label incorporated into the cells as theyproliferate; and (b) determining the extent of proliferation of thecells to identify if the compound is an effective antagonist.
 20. Aprocess for diagnosing a disease or a susceptibility to a diseaserelated to an under-expression of the polypeptide of claim 10comprising: determining a mutation in the nucleic acid sequence encodingsaid polypeptide.